1. Field of the Invention
The present invention relates to vaccines useful for the prevention or modification of microbial pathogenesis. In particular, this invention relates to vaccines comprising genetically attenuated microbial pathogens.
2. Description of the State of the Art
Microbial pathogens, or disease-producing microorganisms, can infect a host by one of several mechanisms. They may enter through a break in the skin, they may be introduced by vector transmission, or they may interact with a mucosal surface. Disease ensues following infection of the host, when the potential of the pathogen to disrupt normal bodily functions is fully expressed. Some disease-producing microorganisms possess properties, referred to as virulence factors, that enhance their pathogenicity and allow them to invade host or human tissues and disrupt normal bodily functions. The virulence of pathogens, that is, their ability to induce disease, depends in large part on two properties of the pathogen, invasiveness and toxigenicity. Invasiveness refers to the ability of the pathogen to invade host or human tissues, attach to cells, and multiply within the cells or tissues of the host. Toxigenicity refers to the ability of a pathogen to produce biochemicals, known as toxins, that disrupt the normal functions of cells or are generally destructive to cells and tissues.
The means by which vertebrates, particularly birds and mammals, overcome microbial pathogenesis is a complex process. Pathogens that invade the host provoke a number of incredibly versatile and protective systems. The first system that is triggered in response to tissue injury or infection is the complex defense mechanism of inflammation. Key to the inflammatory response is the release of various chemicals, such as histamine, from the injured tissue. Histamine causes blood vessels in the region to dilate, thus increasing local blood flow which results in swelling. The tissue fluid, which becomes loaded with extra clotting proteins from the plasma, begins to coagulate and prevents the normal flow of tissue fluid. As a result, the spread of the pathogen or its toxins is greatly slowed and is more or less confined to the area of tissue injury. In addition to this somewhat mechanical slowing of infection, the various chemicals that are released also serve to guide leukocytes or white blood cells toward the site of injury. The general function of leukocytes is to combat inflammation and infection. Some leukocytes, neutrophils and monocytes, are actively phagocytotic and they ingest microbial pathogens and other foreign material. Other leukocytes, lymphocytes, are key elements in the immune response of the body and are discussed in further detail below.
The rapidity of the inflammatory response is proportional to the extent of tissue destruction. Therefore, as an example, a staphylococcal infection, which produces great tissue destruction, is normally quickly confined by the inflammatory response, while streptococcal infections, which are less destructive, elicit a much slower inflammatory response. As a consequence, the confinement of Streptococcus and its toxins is less likely to be successful, and the pathogenic invasion can continue to spread throughout the body.
If all other barriers fail and the microbial pathogen or its toxins penetrate the body's defenses, as discussed above, and reach the bloodstream, the lymphoid tissue of the spleen, liver, and bone marrow will remove and destroy the foreign material as the blood circulates through these organs. Lymphoid tissue is composed primarily of a meshwork of interlocking reticular cells and fibers. Clinging to the interstices of the tissues are large numbers of leukocytes, more specifically lymphocyte cells, and other cells in various stages of differentiation, such as plasma cells, lymphoblasts, monocyte-macrophages, eosinophils and mast cells. The two main lymphocytes, T cells and B cells, have different and complementary roles in the mediation of the antigen-specific immune response.
The immune response is an exceedingly complex and valuable homeostatic mechanism having the ability to create antibodies against any foreign material or every chemical structure that might appear on the surface of a microbial pathogen or one of its toxic products. These foreign materials, referred to as antigens, elicit the ultimate response of the host, the acquired immune system, which operates by means of antibodies. An antibody is said to be specific as it attacks and binds only the antigen that triggered its production, thereby inactivating the antigen. The antigen contains some molecular species, usually protein or glycoprotein, that is not normally present in the host organism. Therefore, microorganismal cell membranes or toxins produced by the microorganism are considered antigenic in the host because they possess molecular species not normally present there.
The process of T cell and B cell development or differentiation describes the maturational events that begin with pluripotential bone marrow stem cells and end with a diverse population of specialized functioning T cells and B cells which mediate the cellular and humoral immune systems, respectively. In mammals, lymphocytes pass through the thymus gland in the throat where they differentiate into T cells, which mediate the cellular immune system. These cells are capable of killing other cells, and so this system is primarily effective against intracellular virus infections, fungi and parasitic worms. T cells also conduct an immune surveillance by watching the body's own cells for those that are developing into cancers. Other lymphocytes pass through the Peyer's patches, the small masses of lymphoid (lymphocyte-bearing) tissues that are distributed around the intestines, where they appear to differentiate into B cells. The B cells, through the production and release of antibodies (including secretory immunoglobulins of the secretory IgA subclass), which are a class of proteins known to mediate neutralization of extracellular bacteria and viruses.
There is a high degree of cooperation and interaction between these two classes of lymphocytes. T cells are first to detect the presence of antigens, and react quickly to bind the antigens with their surface receptor molecules. Once the antigen is bound, the T cell begins to proliferate by rapidly dividing and by producing the monomeric immunoglobulin that is localized on the membrane surface. The antigen-antibody (ag-ab) complexes that form as antigen molecules attach to these surface antibodies are released from the T cell and are then picked up by macrophage cells. The macrophage cells eventually become covered with ag-ab complexes that protrude from the surface with bound antigens facing away from the cells. The macrophage cells then present the antigens to B cells.
When a B cell binds to an antigen which has never been encountered, the cell undergoes repeated divisions to produce multiple clones. This event is considered a primary response. Within this population of "identical" cells, some mature to become antibody factories that release immunoglobulins into the blood. When they are fully mature, they become identified as plasma cells, cells that are capable of releasing about 2,000 identical antibody molecules per second until they die, generally within 2 or 3 days after reaching maturity. During the developmental changes, the plasma cells switch from producing general IgM type antibodies to producing highly specific IgG type antibodies. Other cells within this group of clones never produce antibodies but function as memory cells, which carry the program for the production of a highly specific IgG antibody that will recognize and bind a specific antibody.
As a consequence of the initial challenge by an antigen there are now many more cells identical to the original B cell or parent cell, each of which is able to respond in the same way to the antigen as the original B cell. Consequently, if the antigen appears a second time, it will encounter one of the correct B cells sooner, and since these B cells are programmed for the specific IgG antibody, the immune response will begin sooner, accelerate faster, be more specific and produce greater numbers of antibodies. This event is considered a secondary or amnestic response. FIG. 1 shows a comparison of the antibody titer present as a result of the primary and secondary responses. Immunity can persist for years because memory cells survive for months or years and also because the foreign material is sometimes reintroduced in minute doses that are sufficient to constantly trigger low-level immune responses. In this way the memory cells are periodically replenished.
Following the first exposure to an antigen the response is often slow to yield antibody and the amount of antibody produced is small, i.e., the primary response. On secondary challenge with the same antigen the response is more rapid and of greater magnitude, i.e., the secondary response. Achieving an immune state equal to the accelerated secondary response following reinfection with a pathogenic microorganism is the goal that is sought to be induced by vaccines. Vaccines are basically suspensions of viral, bacterial, or other pathogenic agents or their antigens which can be administered prophylactically to induce immunity.
Attempts to vaccinate are almost as old as man's attempts to rid himself of disease. However, during the last 200 years, since the time Edward Jenner deliberately and systematically inoculated a population with cowpox to avoid a smallpox epidemic, vaccination, at least in parts of the world, has controlled the following nine major diseases: smallpox, diphtheria, tetanus, yellow fever, pertussis, poliomyelitis, measles, mumps and rubella. In the case of smallpox, the disease has been totally eradicated from the world. The impact of vaccination on the health of the world's people is hard to exaggerate. With the exception of safer water, no other modality, not even antibiotics, has had such a major effect on mortality reduction and population growth.
In general, active vaccines can be divided into two general classes: subunit vaccines and whole organism vaccines. Subunit vaccines, are prepared from components of the whole organism and are usually developed in order to avoid the use of live organisms that may cause disease or to avoid the toxic components present in whole organism vaccines, as discussed in further detail below. The use of purified capsular polysaccharide material of H. influenza type b, as a vaccine against the meningitis caused by this organism in humans, is an example of a vaccine based upon an antigenic component. See Parks et al., J. Inf. Dis., 136 (Suppl.):551 (1977), Anderson et al., J. Inf. Dis., 136 (Suppl.):563 (1977); and Makela et al., J. Inf. Dis., 136 (Suppl.):543 (1977).
Classically, subunit vaccines have been prepared by chemical inactivation of partially purified toxins, and hence have been called toxoids. Formaldehyde or glutaraldehyde have been the chemicals of choice to detoxify bacterial toxins. Both diphtheria and tetanus toxins have been successfully inactivated with formaldehyde resulting in a safe and effective toxoid vaccine which has been used for over 40 years to control diphtheria and tetanus. See, Pappenheimer, A. M., Diphtheria. In: Bacterial Vaccines (R. Germanier, ed.), Academic Press, Orlando, Fla., pp. 1-36 (1984); Bizzini, B., Tetanus. Id. at 37-68. Chemical toxoids, however, are not without undesirable properties. In fact, this type of vaccine can be more difficult to develop since protective antigens must first be identified and then procedures must be developed to efficiently isolate the antigens. Furthermore, in some cases, subunit vaccines do not elicit as strong an immune response as do whole organism vaccines due to the lack of extraneous materials such as membranes or endotoxins that may be more immunogenic due to the removal of materials that would otherwise mask the protective antigens or that are immunodominant.
Whole organism vaccines, on the other hand, make use of the entire organism for vaccination. The organism may be killed or alive (usually attenuated) depending upon the requirements to elicit protective immunity. The pertussis vaccine, for example, is a killed whole cell vaccine prepared by treatment of Bordetella pertussis cells with formaldehyde. The bacterium B. pertussis localizes in, and colonizes the epithelial lining of the respiratory tract resulting in a highly contagious respiratory disease, pertussis or whopping cough, of humans with morbidity and mortality rates highest for infants and young children. The colonization, further results in local tissue damage and systemic effects caused in large part by toxins produced by B. pertussis. Manclarck et al., Pertussis. Id. at 64-106. These toxins include endotoxin or lipopolysaccharide, a peptidoglycan fragment called tracheal cytotoxin, a heat-labile dermonecrotizing protein toxin, an adenylated cyclase toxin, and the protein exotoxin pertussis toxin. Vaccination is the most effective method for controlling pertussis, and killed whole-cell vaccines administered with diphtheria and tetanus toxoids (DPT vaccine) have been effective in controlling disease in many countries; Fine et al., Reflections on the Efficacy of Pertussis Vaccines, Rev. Infect. Dis., 9:866-883 (1987). However, due to the large amounts of endogenous products, discussed above, contained in the vaccine many children experience adverse reactions upon injection. Endotoxin, which is an integral component of the outer membrane of the gram-negative organism (as well as all other gram-negative organisms) can induce a wide range of mild to severe side effects including fever, shock, leukocytosis, and abortion. While the side effects associated with pertussis vaccine usually are mild, they may be quite severe. The toxic components present in influenza virus vaccines, however, can induce a strong pyrogenic response and have been responsible for the production of Gufflain-Barre syndrome. Since influenza vaccines are prepared by growth of the virus in chick embryos, it is likely that components of the embryo or egg contributes to this toxicity.
The use of killed vaccines has also been described by Switzer et al., U.S. Pat. No. 4,016,253, who applied such a method in preparing a vaccine against Bordetella bronchiseptica infection in swine. In a technical paper by Brown et al., Br. Med. J., 1:263 (1959), the use of killed whole cells is disclosed for preparing a vaccine against chronic bronchitis caused by Haemophilus influenzae. The use of killed cells, however, is usually accompanied by an attendant loss of immunogenic potential, since the process of killing usually destroys or alters many of the surface antigenic determinants necessary for induction of specific antibodies in the host.
In the case of intracellular pathogens, it is generally agreed that vaccines based on live but attenuated microorganisms (live vaccines) induce a highly effective type of immune response. Ideally, these attenuated microorganisms maintain the full integrity of cell-surface constituents necessary for specific antibody induction yet are unable to cause disease, because they fail to produce virulence factors, grow too slowly, or do not grow at all in the host. Additionally, these attenuated strains should have substantially no probability of reverting to a virulent wild-type strain. Traditionally, live vaccines have been obtained by either isolating an antigenically related virus from another species, by selecting attenuation through passage and adaptation in a nontargeted species or in tissue cultures, or by selection of temperature-sensitive variants. The first approach was that used by Edward Jenner who used a bovine poxvirus to vaccinate humans against smallpox.
Selecting attenuation through serial passages in a nontargeted species is the second approach that has been widely successful in obtaining live vaccines. For example, Parkman et al., N. Engl. J. Med., 275: 569-574 (1966), developed an attenuated rubella vaccine after serial multiplication in green monkey kidney cells. A measle vaccine has been prepared by passaging the virus in chick embryo fibroblasts. Vaccines against, polio, hepatitis A, Japanese B encephalitis, dengue, and cytomegalovirus have all been prepared following similar procedures.
While animal models, and especially monkeys, are useful in developing live vaccines, a large uncertainty as to whether a vaccine is truly apathogenic remains until humans have been inoculated. For example, the Daker strain of yellow fever produced from infected suckling mouse brains induced encephalitis in 1% of vaccinees. Another crucial problem is the possible contamination of the vaccine, by exogenous viruses, during passages in cell culture or in animals, especially in monkeys. In light of the more recent knowledge of the potential danger of viruses that can be transmitted from animals to humans, this choice of developing live vaccines is highly questionable.
In contrast to the somewhat haphazard approaches of selecting for live vaccines, discussed above, modem developmental approaches introduce specific mutations into the genome of the pathogen which affects the ability of that pathogen to induce disease. Defined genetic manipulation is the current approach being taken in an attempt to develop live vaccines for various diseases caused by pathogenic microorganisms. Salmonella is a prime example of a pathogenic microorganism whose various species are the cause of a spectrum of clinical diseases that include acute gastroenteritis and enteric fevers. Salmonella infections are acquired by oral ingestion. The microorganisms after traversing the stomach, invade and replicate in the intestinal mucosal cells, Hornik et al., N. Eng. J. Med., 283:686 (1970). Some species, such as, S. typhi can pass through this mucosal barrier and spread via the Peyer's patches to the lamina propria and regional lymph nodes. Salmonella typhi which only infects man is the cause of typhoid fever and continues to be an important public health problem for residents in the less developed world. The parenteral killed whole cell vaccine, now in use, is moderately effective but causes marked systemic and local adverse reactions at an unacceptably high frequency.
In an effort to develop live vaccines which are safer and elicit a higher immunological response researchers have focused their efforts to developing live vaccines having specific genetic mutations. Curtiss, in his U.S. Pat. No. 5,294,441, discloses that S. typhi can be attenuated by creating deletions (.DELTA.) in either or both the cya (adenylate cyclase) and crp (cyclic 3', 5'-AMP [cAMP] receptor protein) genes. cAMP and the cAMP receptor protein, the products of pleiotropic genes cya and crp, respectively, function in combination with one another to form a regulatory complex that affects transcription of a large number of genes and operons. Consequently, mutating either of these genes results in an attenuated microorganism. Furthermore, microorganisms having single mutations in either the cya or crp genes can not supplement their deficiency by scavenging these gene products from a host to be vaccinated. The crp gene product is not available in mammalian tissues and while the metabolite produced by the cya gene product, cAMP, is present in mammalian cells, the concentrations present in the cells which S. typhi invades are below the concentrations necessary to allow cya mutants to exhibit a wild-type phenotype. See, Curtiss et al., Infect. Immun., 55:3035-3043 (1987).
Since cAMP is present in host tissues at some level, Curtiss et al., stabilized the .DELTA.cya microorganisms by introducing a mutation into the crp gene. Tacket et al., Infect. Immun., 60(2):563-541 (1992), conducted a study with healthy adult inpatient volunteers which revealed that attenuated S. typhi having deletions in the cya and crp genes have the propensity to produce fever and bacteremia (bacteria in the blood).
A similar approach in the attempt to develop live vaccines has been taken by Dr. Stocker. The genes mutated by Stocker produce products which are also not available in host tissues. Stocker, in U.S. Pat. No. 5,210,035, describes the construction of vaccine strains from pathogenic microorganisms made non-virulent by the introduction of complete and non-reverting mutational blocks in the biosynthesis pathways, causing a requirement for metabolites not available in host tissues. Specifically, Stocker teaches that S. typhi may be attenuated by interrupting the pathway for biosynthesis of aromatic (aro) metabolites which renders Salmonella auxotrophic (i.e., nutritionally dependent) for p-aminobenzoic acid (PABA) and 2,3-dihydroxybenzoate, substances not available to bacteria in mammalian tissue. These aro-mutants are unable to synthesize chorismic acid (a precursor of the aromatic compounds PABA and 2,3-dihydroxybenzoate), and no other pathways in Salmonella exist that can overcome this deficiency. As a consequence of this auxotrophy, the aro-deleted bacteria are not capable of proliferation within the host; however, they reside and grow intracellularly long enough to stimulate protective immune responses. In the technical paper authored by Tacket et al., discussed above, attenuated strains of S. typhi were also constructed for use as vaccines by introducing deletions in the aro C and aro D genes, according to Stocker. These attenuated strains administered to healthy inpatient volunteers also have the propensity to produce fever and bacteremia.
Aside from the unacceptable side effects which result from the use of vaccine strains attenuated as a result of mutations in the cya and crp genes and the aro genes, the teachings of Curtiss and Stocker both suffer further limitations of only developing vaccines created by mutating a particular class of genes. That is, Curtiss and Stocker both teach that live vaccines may only be provided for by creating mutations in a class of genes whose products are not present in the host to be vaccinated at the concentrations necessary for the pathogen to proliferate. Consequently, there exists a vast potential for preventing additional diseases by providing live vaccines based on mutations which exist in a class of genes whose products are present in the host, to be vaccinated, at concentrations high enough for the pathogen to proliferate.
There is still a need, therefore, for vaccines that are prepared from live, non-virulent microorganisms, which are safe and when administered to a host will induce a highly effective type of immune response in the host susceptible to disease from corresponding pathogenic microorganisms.